RNA去甲基化酶的作用机制和抑制剂研究

N6-methyladenosine (m6A) has been discovered to be widespread in the transcriptome，playing fundamental biological function on gene regulation. The fact that the function of the obesity-risk factor FTO is to demethylate m6A in mRNA clearly indicates a novel and reversible regulatory mechanism that presents in mammalian cells and stimulates further efforts to explore m6A modifications and related enzymes. In addition, FTO is also reported to be involved in various disease processes, including type II diabetes and cancers. These studies have provided new insights on the forefront of "RNA epigenetics", focusing on the study of "RNA modification/demodification and regulation" in chemical biology. The elucidation of dynamic regulation of mRNA post-transcriptional modifications is just beginning; thus, the physiological function of the ubiquitous m6A post-transcriptional modification remains largely unclear. On the other hand, to our knowledge, no inhibitor has been developed for human nucleic acid demethylases, in particular mRNA demethylase FTO. We have reported our initial steps towards a structure-based in silico high-throughput screening and further structural optimization, thus leading to the identification of the natural product rhein as the first potent FTO inhibitor with cellular activity. On the basis of this solid result, the applicant dedicates to develop more selective inhibitors towards m6A demethylases, which might serve as better tools in the explorations of cellular function of m6A modification as well as the biology of RNA demethylases, and provide target validation and lead compounds for drug discovery. This project aims to publish 2-3 papers, and output 1-2 patents.

mRNA中N6位甲基化腺嘌呤(m6A) 广泛分布于基因转录区，在广谱基因表达中发挥基础性调节作用。FTO是目前已知能够在体外和细胞内调控m6A甲基化水平的氧化去甲基化酶，与肥胖和肿瘤等疾病密切相关。这些前沿进展开辟了以"RNA修饰/去修饰与调控"为核心内容的表观遗传学研究领域的新方向。然而，m6A修饰的功能和作用机理，以及m6A修饰及其相关酶的化学调控等关键科学问题亟需取得突破。申请人在发展首个具有细胞活性的FTO抑制剂基础上，利用化学生物学和分子药理学为主要手段，致力于探索核酸去甲基化酶识别甲基化修饰底物选择性的分子机制研究，开发细胞上具有高选择性的RNA去甲基化酶抑制剂。小分子化合物将充当探针去发现更多的m6A作用蛋白，更全面描述和理解m6A生物学和作用机制，同时为表观遗传相关疾病的药物研究提供潜在新靶标和活性化合物。本课题预期发表高水平论文2-3篇，申请专利1-2项。

mRNA中N6位甲基化腺嘌呤(m6A) 修饰具有广泛的生物学意义，影响mRNA周期的任何环节。FTO是第一个已知的调控mRNA中m6A甲基化水平的氧化去甲基化酶，与肿瘤等疾病密切相关。这些发现开辟了以“RNA修饰/去修饰与调控”为核心内容的表观遗传学研究的新方向。然而m6A修饰及其相关酶的化学调控研究几乎是空白。课题执行人在发展第一个具有细胞活性的FTO抑制剂基础上，致力于探索去甲基化酶识别底物选择性的分子机制研究，开发细胞水平上具有高选择性的小分子抑制剂。 首先揭示了天然产物大黄酸通过不同的分子识别机理分别抑制FTO和AlkB去甲基化的活性。其次在深入研究大黄酸/FTO作用模式和FTO/ALKBH5底物识别机制基础上，通过荧光偏振的高通量筛选方法发现了一个高选择性的FTO抑制剂甲氯芬那酸，揭示FTO结构中疏水性的底物识别盖子结构域是MA实现FTO和ALKBH5间选择性的关键。甲氯芬那酸乙酯（MA2）作为FTO的抑制剂能够抑制神经胶质瘤细胞的增殖，预示着FTO的靶标成药性。此外，合作研究发现，荧光素可以作为FTO的选择性抑制剂示踪FTO的细胞定位。这些重要进展为研究mRNA甲基化修饰及其相关酶的生物学功能提供具有化学本质的小分子工具，同时为RNA表观遗传相关疾病的药物研究提供新靶标和先导化合物。